Yinzhihuang injection induces apoptosis and suppresses tumor growth in acute myeloid leukemia cells

Background The unmet needs in treating acute myeloid leukemia(AML) promote us to look for more effective and less toxic therapies. In this study, we discovered that Yinzhihuang injection(YZHI), a traditional Chinese patent medicine for hepatitis treatment, suppressed the growth of AML cells. Method Anti-proliferative activities of YZHI were measured by CCK-8 assay. Cell cycle arrest was evaluated by PI staining, and apoptosis was evaluated by annexin V/PI staining. To explore the cell cycle arrest and cell death mechanism induced by YZHI, we assessed a series of assays, including measurements of the protein expression and cellular ATP. The anti-tumor activity was further demonstrated in nude mice. Results Flow cytometric and biochemical analysis revealed that YZHI caused cell cycle arrest and induced apoptosis in the AML HL-60 cells. Mechanistically, YZHI activated AMPK by promoting phosphorylation of the kinase. The active AMPK negatively regulated the downstream target mTORC1, leading to the inhibition of cell proliferation and induction of apoptosis. Pretreatment with the AMPK inhibitor compound C rescued YZHI induced apoptosis and partially restored cell proliferation of HL-60. Consistent with the data in vitro, YZHI obviously suppressed subcutaneous xenograft growth in nude mice. Conclusions In a word, our data suggest that YZHI can be repurposed for the treatment of AML, which is worthy of further clinical evaluation.


Introduction
Acute myeloid leukemia (AML), accounting for ~20% of childhood and ~80% of adult leukemia, respectively, is a rapidly progressing hematopoietic malignancy [1,2].It is characterized by the accumulation of clonal myeloid progenitor cells that cannot differentiate into mature blood cells and is accompanied by multilineage cytopenias [3].While it is initially responsive to cytarabine and anthracycline chemotherapy in most patients, the long-term outcomes of AML have not improved significantly for over three decades.Several targeted drugs for AML treatment, including the fms-like tyrosine kinase 3 (FLT3) inhibitors, the mutant isocitrate dehydrogenase (mIDH) inhibitors and the B-cell lymphoma-2 (BCL-2) inhibitors, have been approved in recent years [4][5][6][7].However, the 5-year overall survival rate for patients <60 years remains low, ranging from 35% to 40% [8,9].Therefore, novel therapies are urgently needed for AML management.
Traditional Chinese Medicine (TCM) use medicinal herbs to treat and relieve human diseases.Yinzhihuang injection (YZHI) is an injectable polyherbal prescription derived from the ancient Chinese medicine formula of Yinchenhao decoction.It comprises four herbal extracts, including Artemisia, Gardenia Fructus, Baicalin, and Honeysuckle, and is widely used to treat acute and chronic hepatitis in China [10,11].YZHI alleviates jaundice by promoting bile excretion [12] and attenuates intrahepatic cholestasis by regulating farnesoid X receptor and Mrp2/Bsep [13,14].By assessing mouse T-cell proliferation and cytokine/chemokine produced by human peripheral blood mononuclear cells, Chen [15] showed that YZHI is a potent inhibitor of T-cell activation.Yao [16] demonstrated that YZH could activate the AMPK/SREBP-1 pathway to ameliorate hepatic steatosis and diet-induced obesity.However, no anti-tumor effect of YZHI has been reported so far.
Here, we found the anti-AML effects of YZHI and investigated its underlying mechanisms.The study suggests that YZHI is a promising therapy for AML patients.

Apoptosis assay
HL-60 cells were seeded in 6-well plates at 5×10 5 cells/well and treated with YZHI of indicated concentrations.After 24 h, the cells were collected in the tube, washed with cold phosphatebuffered saline (PBS) (pH 7.4), then incubated with 5 μl PI and 5 μl FITC Annexin V in 100 μl 1×Binding Buffer at room temperature in the dark for 20 min.Next, the cells were resuspended in 400 μl 1×Binding Buffer.Cellular apoptosis was assessed with Annexin V-FITC Apoptosis Detection Kit I(BD Bioscience).The cells were analyzed by flow cytometer (BD Bioscience) after passed through a filter.The data were analyzed by FlowJo 7.6 software.

Cell cycle analysis
HL-60 were seeded at 5×10 5 cells/well in 6-well plates and incubated with medium containing YZHI for 24 h.The next day cells were collected in the tube, washed with cold PBS twice.Then 70% ethanol was added in cold PBS to fix cells for 2 h at -20˚C.Next, cells were stained with 500 μl PI/RNase A (KeyGen BioTECH) at room temperature for 30 min.The cell cycle was analyzed by flow cytometry (BD Bioscience) and FlowJo 7.6 software.

Western blotting
HL-60 cells were seeded at 1×10 6 cells/well in 6-well plates, incubated with medium containing YZHI for indicated time point.Cell lysates were prepared with RIPA buffer supplemented with phosphatase inhibitor (Roche) and protease inhibitor cocktail (Roche).The protein concentrations were quantified by BCA protein assay kit (P0012, Beyotime).Protein samples were heated at 95˚C for 10 min following diluted in 5 x loading buffer.Then, 30 μg of each sample was fractionated on SDS-PAGE gels, transferred to PVDF membrane (Millipore) and incubated with different primary antibodies overnight at 4˚C.Next, HRP-conjugated secondary antibodies(1:10000) were added at room temperature following the membranes were washed with 1×TBST.Proteins were visualized with SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo Scientific).

Cellular ATP measurement
HL-60 cells were plated in 96-well plates at 6×10 4 cells/well in duplicate, incubated with medium containing YZHI for 24 h.ATP concentration was determined with CellTiter-Glo Assay Kit (G7573, Promega).

Mitochondrial membrane potential(MMP) measurement
MMP was determined by JC-1 staining (M34152, Invitrogen).HL-60 cells were seeded at 5×10 5 cells/well in 6-well plates, incubated with medium containing YZHI for 24h.The next day cells were collected in the tube, washed with PBS, then stained with JC-1(2 μM final concentration) in warm medium for 20 min at 37˚C.Next, Cells were resuspended in 500 μl PBS following washed with PBS twice.The cells in different groups were analyzed by flow cytometer (BD Bioscience) after passed through a filter.The data were analyzed by FlowJo 7.6 software.

Xenograft assay
All mice used in the animal experiments were 4-6 week old female nude mice.5×10 6 HL-60 cells were inoculated subcutaneously into both hind limbs of nude mice.We randomly divided the mice into two groups: vehicle (normal saline, ip), YZHI (135.3 mg/kg/d, ip) when tumor volumes reached 100 mm 3 .The therapy lasted for 21 days and the tumor size and body weight were measured every other day.The tumor volumes were calculated following equation: volume = (width 2 × length)/2 [18].All animal studies were approved by the Animal Ethical Committee of Affiliated Hospital of Southwest Medical University.Project codes:20211118-030.Euthanasia was performed by delivering carbon dioxide via compressed gas followed by immediate cervical dislocation.

Statistical analyses
All data were shown as mean ± standard deviation (SD).And statistical analyses were performed through Graph Pad Prism 8.0 software.The data were analyzed by one-way analysis of variance (ANOVA) if comparing the differences more than two groups.Student's t test was selected to compare two groups.* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001.

YZHI inhibits AML cell proliferation
YZHI was first diluted to the highest concentration of 125 μg/ml, corresponding with the maximum clinical doses, to evaluate its cytotoxic effects on cancer cell lines [15].Then, a serial dilution was prepared and used to incubate the cells.As shown in Fig 1A -1C (S1 Fig) , YZHI suppressed the growth of three AML cell lines dose-dependently.The IC 50 of HL-60, THP-1 and KG-1 were 9.6±2.4μg/ml, 33.8±5.9μg/ml and 40.6±7.6 μg/ml, respectively.Furthermore, YZHI did not obviously affect the proliferation of normal cell lines from liver (Lo2) and prostate stroma (WPMY-1), whose IC 50 were over 125 μg/ml (Table 1, S1 Table ).YZHI was also active on ALL cell lines (CEM-C7, Nalm6, SupB15) and breast cancer cell line MDA-MB-231, barely affecting colon cancer cell line HCT116 (Table 1, S1 Table ).So, YZHI exhibited a broad anti-cancer spectrum.We focused on AML cells and did the rest of the studies mainly on HL-60, the most sensitive cell line to YZHI treatment.In accord with the dose effect of YZHI, the growth of HL-60 cells was also inhibited in a time-dependent manner (Fig 1D and S1 Fig).The relative cell viability at 24 h, 48 h and 72 h was approximately 68.1%, 47.1%, and 22.6%.These results suggest that YZHI affects the proliferation of AML cell lines in a dose-and time-dependent manner.The IC 50 of YZHI on various cell lines after 72 h treatment. https://doi.org/10.1371/journal.pone.0289697.t001

YZHI induces cell cycle arrest in HL-60 cells
We further evaluated the effect of YZHI on cell cycle distribution.Flow cytometry analysis after PI staining revealed that YZHI significantly increased the cell percentage in the G1 phase.
In the control group, there was merely 64.39%.But it was increased to 71.45%, 76.75% and 82.4% when YZHI was treated at 8, 24 and 72 μg/ml (Fig

YZHI induces apoptosis in HL-60 cells
As induction of apoptosis is the most common mechanism of chemotherapeutic drugs on cell proliferation, we next explore whether YZHI can induce apoptosis in HL-60 cells.Collectively, we conclude that YZHI induces apoptosis in AML cells from these results.

AMPK/mTORC1 signaling pathway mediates YZHI-induced apoptosis and growth inhibition in HL-60 cells
To understand the underlining mechanism of YZHI-induced apoptosis, we did network pharmacology-based analysis as previously described [20].It implied that the 5' -AMP-activated protein kinase (AMPK) pathway and the cellular energy production might be the priority target of YZHI.To test this possibility, we first measured the intracellular ATP levels in HL-60 cells after YZHI treatment.Indeed, YZHI dose-dependently decreased the ATP levels ( The mammalian target of rapamycin complex 1 (mTORC1), which controls apoptosis, protein synthesis and cell proliferation [22], is a protein complex and negatively regulated by AMPK [23].Therefore, we speculated that YZHI induces apoptosis and inhibits cell proliferation through the AMPK/mTORC1 pathway.Indeed, YZHI inhibited p-P70S6K and p-4E-BP1, the effectors of mTORC1 responsive for protein synthesis (Fig 4B and S4B Fig).
To confirm the activation of AMPK is essential for the anti-cancer effect of YZHI, compound C, which is a specific inhibitor of AMPK, is used to block the signaling [24].We found that compound C partially decreased the apoptosis (Fig 4D , 4E

YZHI suppresses xenograft growth in vivo model
YZHI could inhibit AML cell growth and induce apoptosis in vitro, as shown above, we would explore whether it was still effective in vivo model.HL-60 cells were inoculated subcutaneously into both hind limbs of nude mice, and YZHI was administered at 135.3 mg/kg/d intraperitoneally, which was corresponding to the maximum dose of YZHI injected intravenously in human conversion guideline and the clinic following the animals [25].There was not a significant difference in body weight between two groups.(Fig5A

Discussion
Despite advances in therapeutic strategies, there is still a high resistance to the standard treatment of AML [26].The characteristic with multiple components and multiple targets of TCM provides an alternative regimen for the treatment of AML.YZHI is a traditional Chinese patent medicine for the treatment of acute and chronic hepatitis.It has been used in the clinic for twenty years, and the overall safety has been approved.In line with this point, we observed that YZHI didn't obviously affect the proliferation of normal cell lines from Live (Lo2) and prostate stroma (WPMY-1) (Table 1, S1 Table ).For the first time, our study reported the anticancer activities of YZHI both in vitro and in vivo and provided the rationality for repurposing the drug for AML treatment.AMPK, which monitors the bioenergetic state, is a highly conserved Ser/Thr protein kinase complex [21].mTORC1 is one of the major downstream targets negatively regulated by AMPK [27].The signaling axis plays a critical role in tumorigenesis and thus an attractive target for cancer treatment [28][29][30].AMPK activator metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) have been shown promising in treating colon cancer, hepatocarcinoma, lymphoma and prostate cancer through inhibition of mTORC1 [31][32][33][34].In this study, we showed that YZHI induced apoptosis and inhibited the proliferation of AML cells by the regulation of the AMPK/mTORC1 signaling pathway (Fig 6).On the other hand, our study may also provide mechanistic insights of YZHI on hepatitis, in which the AMPK/ mTORC1 signaling pathway also plays an essential role [35].
The most abundant compound in YZHI is baicalin, which is from Huangqin [36].In line with our findings, activating the AMPK/mTOR signaling pathway, baicalin suspresses the migration and proliferation of human non-small cell lung carcinoma cells [37].In addition, YZHI contains quercetin, luteolin, kaempferol, beta-sitosterol, stigmasterol and isorhamnetin, which have been shown to induce tumor cell apoptosis through the AMPK/mTORC1 signaling pathway [38][39][40][41].Future studies are needed to compare these compounds' effect on the activity of the AMPK/mTORC1 pathway and the anti-cancer efficiency.
In recent decade, some serious adverse drug reaction have raised concerns about the potential toxicity and safety related to TCM injections [42].Compared with other formulations, TCM injections have advantages of excellent drug-likeness, including good bioavailability and solubility [43].In addition, TCM injections have been widely used in acute or severe diseases due to the rapid and accurate curative effect.Therefore, we believe that TCM injections provide valuable sources for research and application of drugs.YZHI can be reformulated with better quality control properties and less complexity, or active ingredients can be identified from YZHI to develop new drugs against AML.

Fig 1 .
Fig 1. YZHI inhibits AML cell proliferation.(A-C) HL-60, KG-1 and THP-1 cells were treated with different concentrations of YZHI for 72 h.YZHI was first diluted to the highest concentration of 125 μg/ml and then twice diluted in turn.(D) HL-60 cells were treated with 24 μg/ml YZHI for various times.Cell viability (% of control) was performed by CCK-8 assay.(mean ± SD, n = 3 biologically independent samples).https://doi.org/10.1371/journal.pone.0289697.g001 2A, 2B and S2A Fig).Furthermore, in line with causing the cells arrested in the G1 phase, YZHI treatment downregulated the levels of Cyclin D1, an essential regulator of cell cycle progression (Fig 2C and S2B Fig).Taken together, these data suggest that YZHI induces cell cycle arrest in HL-60 cells.
YZHI significantly increased the percentage of apoptotic cells, as shown in Fig 3A (S3A Fig).In the control group, there was merely 2.65% of apoptotic rate.But it was remarkably increased to 22.89%, 41.2% and 57.9% when YZHI was treated at 8, 24 and 72 μg/ml (Fig 3A, 3B and S3A Fig).The decrease of MMP is a detection index in the early phase of apoptosis.Therefore, we checked MMP with JC-1 staining and discovered that YZHI significantly induced the reduction of MMP in HL-60 cells (Fig 3C, 3D and S3A Fig).Accordingly, well-known apoptosis markers [19], such as cleaved PARP and cleaved Caspase-3, were induced by YZHI dosedependently (Fig 3H and S3B Fig).In addition, the antiapoptotic protein Bcl-2 was decreased and the proapoptotic protein Bax was significantly induced with YZHI treatment in HL-60 cells (Fig 3H and S3B Fig).Furthermore, pretreatment with the pan-caspase inhibitor